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  1. NTU Theses and Dissertations Repository
  2. 生物資源暨農學院
  3. 食品科技研究所
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34354
完整後設資料紀錄
DC 欄位值語言
dc.contributor.advisor吳瑞碧(James Swi-Bea Wu)
dc.contributor.authorShu-Chin Yangen
dc.contributor.author楊舒秦zh_TW
dc.date.accessioned2021-06-13T06:04:30Z-
dc.date.available2011-06-27
dc.date.copyright2006-06-27
dc.date.issued2006
dc.date.submitted2006-06-16
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34354-
dc.description.abstract靈芝在中國傳統醫學中一直佔有極崇高的地位,被視為是滋補強壯、扶正固本的珍貴藥材。本實驗以豆科植物為發酵基質之靈芝發酵液為實驗材料,由細胞實驗篩選出具有免疫活性之靈芝發酵液樣品,進入動物實驗以評估體內非特異性免疫調節作用;另將酒精沈澱後之靈芝多醣刺激人類單核細胞觀察細胞增生能力,並以蛋白質體學探討受靈芝多醣刺激後之免疫細胞內蛋白質表現的變化。實驗結果顯示,靈芝發酵液GL-93-12-21具有促進人類單核細胞分泌細胞激素的能力,並對人類白血病細胞U937產生生長抑制之效果,且其酒精沈澱多醣能活化巨噬細胞RAW 264.7分泌NO及TNF-α,根據多醣分析結果此靈芝多醣特性與文獻指出之活性多醣特徵相符。經動物實驗觀察體內非特異性免疫調節反應,此批靈芝發酵液具有促進小鼠抗體IgG之生成、促進脾臟細胞中T及B淋巴細胞之增生、增加細胞激素TNF-α、IL-2及IFN-γ分泌量、活化單核細胞吞噬能力及促進自然殺手細胞活性之功效。另實驗將靈芝多醣刺激人類單核細胞後觀察到細胞增生現象,並以蛋白質體學研究細胞內蛋白質表現的變化,結果共有19個表現量差異性在50 % 以上之蛋白質點經由質譜儀(MALDI-Q-TOF)被鑑定出,除了一個為表現量下降外,其餘皆為表現量上升之蛋白質點。經資料庫比對及蛋白質功能鑑定分析結果顯示,細胞骨架及其結合蛋白與核糖核酸蛋白表現量的上升,推論與靈芝多醣刺激免疫細胞的活化及增生有相關。此外,annexin A6及伴護子(chaperone)表現量的增加,可能對於B淋巴細胞的活化與其膜上接受器之表現有所影響。zh_TW
dc.description.abstractGanoderma lucidum is one of the most well known Chinese medicine in ancient times and is considered as the most valuable plant for health and longevity promotion. In this study, we investigated the effects of Ganoderma lucidum liquid fermentation broth on immunomodulatory activities in vitro and in vivo. After an in vitro experiment, we selected the most active fermentation product for experiments on non-specific immune response in BALB/c mice. In addition, we observed the MNC proliferation effect and performed the proteomic analysis of the protein expression by human mononuclear cells (MNC) treated with ethanol precipitated Ganoderma lucidum polysaccharide. Results show that Ganoderma lucidum fermentation product GL-93-12-21 stimulates human mononuclear cells to secrete cytokines and inhibit the human myelocytic lymphoma U937 cell growth. Moreover, the ethanol precipitated polysaccharide stimulats NO and TNF-α production in RAW 264.7 macrophage, and the polysaccharide characteristics are similar with bioactive polysaccharides that have been reported. In vivo studies showed that oral administration of Ganoderma lucidum fermentation product GL-93-12-21 on BALB/c mice significantly (P<0.05) increases the secretion of IgG, the proliferation of T- and B-lymphocyte, the level of TNF-α、IL-2 and IFN-γ production, the phagocytosis effect of monocytes and the cytotoxicity of nature killer cells. Results also show that Ganoderma lucidum polysaccharide stimulates the proliferation of human mononuclear cells and changes the protein expression. There are 19 protein spots (1 down-regulated and 18 up-regulated) differentially expressed more than 1.5-fold, which was identified by quantitative image analysis and matrix assisted laser desorption ionization-quadrupole-time of flight (MALDI-Q-TOF) mass spectrometry. The Swiss-Prot database search indicated that increased expression of cytoskeleton and ribonucleoprotein is associated with the immune cell activation and proliferation. Furthermore, the increase of annexin A6 and chaperon protein expression, may play a part in activating of B lymphocyte and assembling of B cell receptor.en
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dc.description.tableofcontents目錄 Ⅰ
圖表目錄 Ⅴ
中文摘要……………………………………………………………… .1
英文摘要………………………………………………………………..2
第一部份 以豆科植物為發酵基質之靈芝發酵液免疫活性分析…....5
第一章 前言…………………………………………………………....6
第二章 文獻整理………………………………………………………7
一、靈芝的歷史地位……………………………………………..7
二、靈芝之分類與形態…………………………………………..7
三、靈芝的生理功能……………………………………………..8
四、靈芝多醣與免疫調節之關係………………………………..12
五、靈芝的栽培..............................................................................14
六、發酵基質介紹..........................................................................17
(一)黑豆..............................................................................17
(二)黃耆..............................................................................18
七、免疫反應……………………………………………………..20
(一)非特異性免疫反應…………………………………..20
(二)特異性免疫反應……………………………………..22
(三)細胞激素……………………………………………..23
八、健康食品……………………………………………………..24
第三章 材料與方法…………………………………………………....26
第一節 靈芝發酵液體外免疫活性分析............................................26
一、實驗材料……………………………………………………..26
二、實驗方法……………………………………………………..28
(一)靈芝發酵液之前處理………………………………....28
(二)人類白血病細胞U937之培養……………………….28
(三)人類單核細胞條件培養液(MNC-CM)之製備…...28
(四)靈芝發酵液間接抑制U937生長之試驗…………….30
(五)分析單核細胞條件培養液(MNC-CM)中細胞激素
的分泌………………………………………………..31
(六)老鼠巨噬細胞RAW 264.7之培養…………………...32
(七)靈芝多醣刺激老鼠巨噬細胞株RAW 264.7分泌
一氧化氮之測定………………………………………33
(八)靈芝多醣刺激老鼠巨噬細胞株RAW 264.7分泌
TNF-α之測定…………………………………………34
第二節 靈芝發酵液對BALB/c小鼠體內非特異性免疫調節實驗…35
一、實驗架構………………………………………………………36
二、實驗材料………………………………………………………37
三、實驗方法………………………………………………………38
(一)動物飼養……………………………………………….39
(二)實驗組別……………………………………………….39
(三)分離鼠脾臟細胞……………………………………….39
(四)脾臟細胞增殖反應…………………………………….40
(五)抗體分泌實驗…………………………………………..41
(六)細胞激素分泌實驗…………………………………….42
(七)表面標記分析………………………………………….44
(八)自然殺手細胞活性分析……………………………….45
(九)吞噬細胞活性………………………………………….46
(十)統計分析……………………………………………….47
第四章 實驗結果………………………………………………………48
第一節 體外免疫活性分析實驗(in vitro)……………………….48
一、靈芝發酵液間接抑制U937細胞生長之實驗………………..48
二、分析單核細胞條件培養液(MNC-CM)中細胞激素的分泌..49
三、靈芝多醣刺激老鼠巨噬細胞株RAW 264.7分泌一氧化氮
(NO)與細胞激素TNF-α之能力……………………………49
第二節 體內非特異性免疫調節實驗(in vivo)……………………50
一、體重變化………………………………………………………50
二、組織重量………………………………………………………50
三、血清中IgG抗體含量及變化量……………………………….51
四、脾臟細胞增生能力……………………………………………52
五、鼠脾臟細胞條件培養液中細胞激素的分泌…………………52
(一)細胞激素TNF-α含量…………………………………52
(二)細胞激素IL-2含量……………………………………53
(三)細胞激素IFN-γ含量…………………………………..53
六、血液中單核球之吞噬能力……………………………………53
七、脾臟細胞表面抗原分析………………………………………54
八、自然殺手細胞活性分析………………………………………55
第五章 討論………………………………………………………………56
第六章 結論………………………………………………………………63
第二部分 靈芝多醣處理人類周邊血單核細胞之蛋白質體學探討....81
第一章 前言……………………………………………………………82
第二章 文獻整理………………………………………………………83
一、蛋白質體學(proteomic)之介紹..........................................83
二、蛋白質體技術..........................................................................84
(一)蛋白質的分離................................................................84
(二)蛋白質的鑑定................................................................86
第三章 材料與方法……………………………………………………90
第一節 靈芝發酵液經酒精沈澱之多醣刺激人類單核細胞增生....90
一、實驗材料..................................................................................90
二、實驗方法……………………………………………………..91
第二節 以蛋白質體學分析靈芝發酵液經酒精沈澱之多醣處理
人類單核細胞........................................................................92
一、實驗材料..................................................................................92
二、實驗方法……………………………………………………..94
(一)單核細胞蛋白質萃取液之製備....................................94
(二)蛋白質萃取液之定量…………………………………95
(三)二維電泳分離蛋白質…………………………………96
1. 第一維等電焦集電泳………………………………….96
2. 第二維聚丙烯醯胺膠體電泳………………………..97
3. 膠片體染色………………………………………….....98
(四)蛋白質的鑑定………………………………………..98
1. 膠片掃瞄……………………………………………...98
2. 軟體比對……………………………………………...98
3. 蛋白質膠內水解(in-gel digestion)………………...99
4. 質譜分析與資料庫搜尋………………….………….100

第四章 實驗結果…………………………………………………….101
第一節 靈芝多醣刺激人類單核細胞增生.....................................101
第二節 以蛋白質體學分析靈芝多醣處理人類單核細胞.............101
一、二維電泳(2-DE)分離細胞蛋白質...................................101
二、電泳膠片影像比對分析……………………………………102
三、蛋白質身份鑑定與其功能性之探討………………………103
第五章 討論…………………………………………………………..105
第六章 結論…………………………………………………………..115
總結……………………………………………………………………135
參考文獻………………………………………………………………149
dc.language.isozh-TW
dc.title靈芝發酵液之免疫活性分析及以靈芝多醣處理人類單核細胞之蛋白質體學探討zh_TW
dc.titleImmunomodulatory Activities of Ganoderma lucidum Fermentation Products and Proteomic Analysis of Human Mononuclear Cells Following Treatment with Ganoderma lucidum Polysaccharideen
dc.typeThesis
dc.date.schoolyear94-2
dc.description.degree碩士
dc.contributor.oralexamcommittee陳水田(Shui-Tein Chen),江啟銘(Chii-Ming Jiang),廖慧芬(Hui-Fen Liao),許輔(Fuu Sheu)
dc.subject.keyword靈芝,免疫調節,人類單核細胞,蛋白質體學,zh_TW
dc.subject.keywordGanoderma lucidum,Immunomodulatory,Human mononuclear cells,Proteomics,en
dc.relation.page157
dc.rights.note有償授權
dc.date.accepted2006-06-16
dc.contributor.author-college生物資源暨農學院zh_TW
dc.contributor.author-dept食品科技研究所zh_TW
顯示於系所單位:食品科技研究所

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